In early stage, microscopic technology is realized mainly by electron microscope and optical microscope. An optical microscope uses optical lenses to magnify images of a sample to reach a resolution up to 200 nm. On the other hand, an electron microscope utilizes the short wavelength characteristic of electrons to observe micro crystal structure or biological cellular structure. In the case of a transmission electron microscope, a resolution up to 0.1 nm can be reached. However, the transmission electron microscope would disadvantageously cause chemical changes in the sample; and electron beams illuminating the samples tend to destroy organic materials, molecular structures, DNA, etc.
A confocal microscope is developed from the optical microscopic technology. It has a light source that emits laser light, which passes lenses to focus on the sample. If the sample is located at the focal point, reflected light would pass the lenses to condense on the light source and form a confocal condition. The confocal microscope includes a dichroic mirror arranged on the optical path of the reflected light for refracting the reflected light to other directions, and a pinhole is provided on a focal point of the dichroic mirror. When the reflected light passes through the pinhole, an image can be formed on a photosensor. When the light source scans the sample, a three-dimensional (3D) image of the sample can be created. U.S. Patent Publication No. 2009/0310083 discloses an application of the confocal microscope using polarized light. Taiwan Patent Publication No. 200619673 also discloses a method of increasing the measuring resolution in depth of a confocal microscope using light interference. U.S. Pat. No. 5,804,813 discloses the use of a differential confocal microscopy, in which a He—Ne laser or a solid-state laser is used as a light source to reach a resolution in depth of 2 μm and a lateral resolution of 0.3 μm. However, the confocal microscope has a main disadvantage of uneasy to show the image quickly. When the confocal microscope is applied in the study of biofilm, collagen protein, cell movement, liquid interface or liquid-gas interface characteristics and the like, there would be considerable limitation. Moreover, the confocal microscope has a resolution in depth being limited to the size of the pinhole and accordingly has very small penetration depth, making the confocal microscope not good enough for use.
As to tomographic imaging, such as the optical coherence tomography (OCT) imaging, it can penetrate the sample to directly observe the interior of the sample. For example, the OCT imaging can penetrate deep into the bio-tissue by several millimeters and has a resolution in depth of 15-20 μm or even higher. Since the OCT imaging has high resolution power, high sensitivity, and three-axis positioning ability, it has particular potential in medical diagnosis. In addition, due to the economical system development cost thereof, the OCT imaging has gained wide attention of users. In the OCT imaging, the depth-scan thereof is achieved by adjusting a reference mirror, and the lateral-scan is achieved by moving the sample laterally or scanning by probing beam, so that backward scattering of light from the sample and the light path of light reflected from the reference mirror overlap with each other in length to form interference. In brief, the OCT imaging has an important advantage that the resolution in depth is independent of the lateral resolution, and the resolution in depth would not be sacrificed due to the increased lateral resolution. Therefore, the OCT imaging is very suitable for forming 3D images. If it is desired to have an increased resolution in depth, it is better to select a light source with a spectral bandwidth as wide as possible. For example, Taiwan Patent Publication No. 200426397 discloses the use of white light-emitting diode to excite fluorescent powder, so as to produce a bluish-purple light source having a relatively wide spectral bandwidth; Taiwan Patent Publication No. 200937005 discloses the use of ultra-wideband optic fiber laser and spectrum modulation system in cross-section analysis of the interior of a semiconductor wafer. Or, for example, U.S. Pat. No. 6,713,742 discloses the disposition of a sample at a specific angle and the use of angled-dual-axis scanning, bringing the sample to move relative to the light source in order to obtain increased resolution.
In view that the confocal microscope and the OCT imaging have their own advantages, Taiwan Patent No. 569008 discloses a real-time multiplex photoelectric sensing system that can be switched between a confocal microscope and an OCT microscope for observing the sample. National Science Council of Executive Yuan, R.O.C. discloses in its research project No. NSC94-2215-E-007-015 a full-field optical coherence microscopy (OCM), in which a high numerical aperture (NA) objective lens is used to provide an effect similar to that provided by a confocal microscope; and the heterodyne interference technique based on the Michelson interferometer is used to combine the optical configuration of the Michelson interferometer with the optical configuration of the confocal microscope. The above structures are used with a scanner mirror and a translation stage to achieve the purpose of obtaining stratified images of a sample. Japanese Patent Publication No. 2007-086428 discloses a confocal microscope system 91, which applies the OCT technique to the confocal microscope. Please refer to FIG. 5. When light L emitted from a light source 92 enters a dividing means 93, the light L is divided into two parts L1 and L2, which enter a confocal optical system 910 and a light modulator 920, respectively. The light L1 entering the confocal optical system 910 sequentially passes an optical fiber 911 and a focusing objective lens 913 to illuminate a sampling object S. The confocal optical system 910 condenses light reflected from the sampling object S to produce catoptric light L3. Meanwhile, the light L2 is adjusted by the light modulator 920 to a desired frequency to form reference light L2. The catoptric light L3 having been interfered by the reference light L2 forms an interference light L4 to pass through an interference detector 96, and an image processor 98 collects interference images of the sample S at different depths. The processed images are displayed on a screen 950. While the combination of the confocal microscope with the OCT microscope can provide images at different depths and having increased resolution, it is still difficult to get 3D images of highly improved resolution when a light source with insufficient bandwidth for lateral scanning is used.
In view of the disadvantages in the prior art, it is desirable to develop an imaging technique that combines the OCT imaging and the confocal imaging to reconstruct an integrated functional 3D image while using the same light source to form high-resolution microscopic images for further produce a high-resolution 3D microscopic image of a sample.